Is 2D HPLC the Answer for You

Importance of the Topic

Two-dimensional liquid chromatography (2D-LC) addresses the limitations of single-dimension separations by significantly increasing peak capacity and orthogonality. This capability is crucial for analyzing highly complex or closely related compounds, confirming purity of target analytes, profiling impurities, and performing online cleanup in pharmaceutical, biopharmaceutical, food and environmental laboratories.

Study Objectives and Overview

This application note series demonstrates 2D-LC workflows and hardware configurations to:

• Introduce basic concepts of heart-cutting and comprehensive 2D-LC.
• Compare separation performance for diverse sample types (taxanes, peptides, beer components, oligonucleotides, monoclonal antibodies, green tea, chiral drugs).
• Showcase method optimization strategies including shifted gradients and high-resolution sampling.
• Highlight benefits of online desalting and automated fraction transfer.

Methodology and Instrumentation

All experiments employed the Agilent 1290 Infinity II 2D-LC platform integrated with OpenLab ChemStation or MassHunter software and LC Image for data visualization. Core components:

• 1290 Infinity II 2D-LC valve module with fixed-volume loops for fraction transfer.
• Agilent binary/quaternary pumps for 1st and 2nd dimensions.
  • 1st dimension: pumps matched to separation mode (RP, IEX, SEC, HILIC, IP-RP, WCX).
  • 2nd dimension: high-pressure binary pump for fast gradients.
• Column sets:
  • RP: Poroshell SB-C18/Aq, EC-C18, Phenyl-Hexyl, PFP.
  • IEX/WCX/Bio: AdvanceBio Bio MAb, Bio SCX, Bio WCX.
  • HILIC: Poroshell HILIC/Z, AdvanceBio HILIC-Z.
  • SEC: Bio SEC-3/5/300, ProSEC 300S.
  • Chiral: Poroshell Chiral-V, ‑T, ‑CD, ‑CF.
• Detectors: Diode-array, QTOF MS, ELSD as required.

Main Results and Discussion

• Taxanes from Taxus extract: Comprehensive RP×RP with shifted 2nd-dimension gradient fully resolved paclitaxel from matrix interferences.
• Beer components: SEC–RP, IEX–RP and RP–RP heart-cutting modes differentiated sugars, proteins, polyphenols in two Japanese beers.
• Peptide mapping: HILIC×RP provided orthogonal separation of oxidized vs native trastuzumab tryptic peptides, enhancing sequence coverage.
• Oligonucleotide analysis: Single heart-cutting enabled online desalting of high-salt samples followed by IP-RP separation of 14–21 mers without offline cleanup.
• mAb charge variants and glycoforms: Multiple heart-cutting 2D-LC coupled with desalting and QTOF detected low-abundance C-terminal lysine variants in a rituximab biosimilar.
• Green tea quantification: High-resolution sampling 2D-LC measured caffeine and (–)-epigallocatechin gallate in coeluting zones with improved accuracy.
• Chiral drug impurity profiling: Online heart-cutting separated racemic ibuprofen impurities in 1D and routed the main fraction to a chiral column in 2D for enantiomeric excess determination with high precision.

Benefits and Practical Applications

2D-LC offers:

• Substantially increased separation space and peak capacity without excessively long gradients or ultra-small particles.
• Enhanced resolution of coeluting or structurally similar compounds.
• Automated fraction transfer for impurity profiling, cleanup and enrichment.
• Flexible coupling of orthogonal modes (RP, IEX, HILIC, SEC, IP-RP, chiral) to tackle diverse analytical challenges.
• Compatibility with MS and UV detection for structural elucidation and quantitation.

Future Trends and Potential Applications

Advances may include integration with high-throughput mass spectrometry, AI-driven method development, expanded column chemistries tailored for emerging biotherapeutics, and miniaturized 2D-LC for point-of-care or field analyses. Continued software innovations will further streamline data visualization, peak tracking and automated decision making.

Conclusion

Two-dimensional liquid chromatography provides a versatile, powerful solution for complex separations across pharmaceutical, biopharmaceutical, food and environmental fields. By leveraging heart-cutting, comprehensive and high-resolution sampling modes, analysts can achieve higher peak capacity, selectivity and throughput while automating purification, impurity profiling and online cleanup.

Reference

• Li D., Schmitz O. J. Use of shift gradient in the second dimension to improve the separation space in comprehensive two-dimensional liquid chromatography. Anal. Bioanal. Chem. 405, 6511–6517 (2013).